Transcription can have a strong stimulatory effect on both recombination and mutagenesis, effects that have been linked to enhanced DNA damage. In the case of transcription-associated mutagenesis, we have found that apyrimidinic/apurinic (AP) sites accumulate to a very high level in highly-transcribed yeast DNA. Most of these AP sites are generated by the enzymatic removal of uracil, and genetic studies demonstrate that the transcription-associated uracil is derived from direct incorporation rather than from cytosine deamination. The enhanced incorporation of uracil suggests that there is a transcription-associated perturbation in the nucleotide pool available to DNA polymerases, and the proposed experiments are designed to further examine the relationship between transcription and the fidelity of DNA synthesis. The first aim will address whether the uracil in highly-transcribed DNA is derived solely from the direct incorporation of dUTP, or whether the UTP used in RNA synthesis can also replace dTTP during DNA synthesis. While genetic data suggest that high transcription of a reporter gene does not elevate uracil incorporation into an unlinked gene, Aim 2 will examine whether the enhanced incorporation of uracil is strictly limited to highly-transcribed DNA, or whether it might extend into adjacent regions. The transcription-associated incorporation of uracil could occur during normal DNA replication, or it could specifically be associated with the more limited DNA synthesis that accompanies DNA repair processes. This issue will be addressed in Aim 3, where cell-cycle regulated promoters will be used to examine whether transcription-associated uracil incorporation is limited to S phase or whether it occurs throughout the cell cycle. Finally, Aim 4 is designed to explore the basis of the very distinctive strandedness exhibited by the repair pathways that remove the AP sites that accumulate in highly-transcribed DNA. Together these experiments will greatly enhance our understanding of a very novel source of mutagenesis that is directly linked to the level of gene expression, and that likely modulates global mutation rates and patterns. PUBLIC HEALTH RELEVANCE: Mutations provide the raw material for evolutionary processes and are causative in a number of human diseases, especially cancer. The proposed experiments use yeast as a model system to explore a novel type of mutagenesis that is specifically linked to transcription. These studies will provide insight into mechanisms that promote targeted mutation accumulation.